In the fruitfly Drosophila melanogaster, neurogenesis is under the control of several loci, including Enhancer of split, whose products appear to determine the fate of neuroectodermal cells during the embryo development. These genes appear to play significant roles in spermatogenesis in addition to neurogenesis. The cDNAs encoding the enhancer of split groucho (ESG) and its related amino-terminal (AES) proteins were isolated and sequenced from human, mouse and Xenopus. The ESG proteins consist of 771 amino-acids, while the AES proteins contain 197 amino-acids. The AES proteins were found to exhibit approximately 50% identity to the amino-terminal region of Drosophila enhancer of split groucho protein. Human AES and two ESG genes have been mapped using human and Chinese hamster hybrid cell lines to chromosomes 19, 9 and 15, respectively. Human AES gene was further localized by fluorescence in- situ hybridization to chromosome band 19p13.3, and its protein-encoding sequence is interrupted by six introns. The evolutionary relationships among these AES and ESG genes were analyzed. The physiological function(s) of vertebrate AES and ESG proteins during spermatogenesis, oogenesis, and embryogenesis, including neurogeneis, are being investigated.